Parathyroid hormone-related protein (PTHrP) is an important regulator of the proliferation and differentiation of both chondrocytes and osteoblasts. This project will explore the molecular mechanisms of PTHrP action during bone development. It will also characterize the genes associated with differentiation of chondrocytes and will characterize the differentiation of cells of the osteoblast lineage in vivo. PTHrP suppresses the expression of Runx2 in chondrocytes. Aim IA will analyze the role of this suppression in mediating the action of PTHrP to delay the differentiation of chondrocytes. Indian hedgehog (Ihh) stimulates the synthesis of PTHrP in chondrocytes. To determine whether the action of Ihh to stimulate PTHrP expression is a direct action on PTHrP-producing chondrocytes, the expression of PTHrP in chondrocytes missing smoothened will be analyzed in chimeric growth plates in Aim IB. To identify genes mediating the action of PTHrP, microarray analysis of E14.5 mouse metatarsals after brief PTH treatment will be performed, and the roles of select genes will be analyzed in Aim 1C. To characterize the genetic changes associated with differentiation of chondrocytes, microarray analysis of RNA amplified from discrete groups of chondrocytes will be conducted in Aim ID, and the roles of select genes will be analyzed. The early stages of differentiation of osteoblast lineage cells and the proliferative capacity of cells at these stages are poorly understood in vivo. In Aim II, site-specific recombination will be used, with promoters active in early stages of osteoblast development, to study the properties of such cells. Conditionally active recombinases will be used to mark discrete cell populations and determine the self-renewing capacity of these cells both in fetal life and adulthood. Microarrays will be used to characterize genes involved in early steps of the osteoblast lineage. The role of the PTH/PTHrP receptor during fetal osteoblast development will be assessed using a floxxed allele of the PTH/PTHrP receptor.